Sigma receptors are saturable, high affinity binding sites for several important classes of psychotropic drugs, including typical antipsychotic, antidepressant, anticonvulsant, and psychotomimetic compounds. They are likely to contribute to the beneficial and/or side-effect profile of these compounds. Sigma sites are present not only in the central nervous system but also occur in several peripheral tissues, and are expressed in high density in a number of tumor cell lines. Furthermore, they are more highly expressed in rapidly proliferating cells than in quiescent cells. They may thus play an important role in cell function. Our studies have shown that agonists at sigma-2 receptors induce morphological changes, followed by apoptotic cell death. These effects were found to occur in various tumor cell lines, as well as in primary cultures of neuronal cells. Sigma-2 agonists induce both a transient and a sustained rise in intracellular calcium levels in human SK-N-SH neuroblastoma cells, which may play a role in cell proliferation or in the induction of apoptosis. The calcium is released from intracellular stores, and is apparently independent of IP3. Furthermore, sigma-2 receptor-induced apoptosis in breast tumor cell lines was found to occur by a novel pathway which was both p53- and caspase-independent. In the last reporting period, we investigated the involvement of sphingolipids in the apoptotic pathway induced by sigma-2 agonists. Ceramides and lysosphingolipids are involved in regulation of cell proliferation. Sigma-2 agonists were found to induce a rise in ceramide and sphingosylphosphorylcholine (a lysosphingolipid) with a concomitant decrease in sphingomyelin. This was not due to activation of sphingomyelinase, but appears to be due to an increase in sphingolipid-ceramide N-deacylase (SCDase)-like activity. In the current reporting period, we continued to investigate the role of sphingolipids in sigma-2 receptor signaling. We have begun to assess the effect of ceramides and various lysosphingolipids on the growth of neuroblastoma cells in culture. Lysosphingolipids have been shown to have various effects on cells and act as both second messengers and as agonists at G-protein coupled receptors. The localization of sigma-2 receptors may also reflect the role of sphingolipids in the signaling mechanism. We have demonstrated in this period that sigma-2 receptors are localized in membrane lipid rafts. Lipid rafts are sphingolipid- and cholesterol-rich segments of membrane which exist as phase-separated domains. These lipid rafts are known to be detergent-resistant, have low buoyant density, and contain lipid-modified proteins such as glycosylphosphatidylinositol-anchored proteins and N-acylated proteins. They also form loci for cell signaling. Several enzymes of sphingolipid metabolism have been localized to membrane lipid rafts. Furthermore, we previously showed that sigma-2 receptors are relatively resistant to solubilization by CHAPS and Triton X-100. We also showed that sigma-2 receptors can stimulate changes in sphingolipids in cell-free Triton X-100 extracts, indicating that signaling remains intact upon detergent treatment. Rat liver membranes were treated on ice with Triton X-100 and the extract subjected to sucrose density gradient centrifugation. Fractions were then analyzed for flotillin-2, a marker for lipid rafts in liver, using Western blotting. Fractions were also assayed for sigma-1 receptor binding activity using [3H](+)-pentazocine and for sigma-2 receptor binding activity using [3H]1,3-di-o-tolylguanidine, in presence of a sigma-1 receptor blocker. Sigma-2 receptors were found to discretely co-localize with flotillin-2. However, sigma-1 receptors were found throughout the gradient. Thus, sigma-2 receptors are resident in membrane lipid rafts. This is consistent with a role in sphingolipid signaling, where sphingomyelin would be readily available as a substrate for a sigma-2 receptor-activated SCDase-like enzyme. We have continued our program of sigma receptor ligand development and characterization. In collaboration with Dr. Andrew Coop (University of Maryland), we characterized a series of N-arylpiperidines as sigma receptor ligands. We had shown that AC927 (N-phenethyl piperidine) had high sigma-1 affinity and moderate sigma-2 affinity. AC927 exhibited antagonist activity, attenuating sigma-2 receptor agonist-induced rises in calcium, formation of ceramide, decrease in sphingomyelin, and cytotoxicity. Several analogs of AC927 were examined, including analogs with 3- and 4-methyl substitution on the piperidine ring, tetrahydroisoquinoline analogs, and (2-pyridyl)piperazine analogs. In each series, N-phenethyl derivatives were compared to N-phenylpropyl derivatives. At sigma-1 receptors, N-phenethyl and N-phenylpropyl derivatives generally had comparable affinities. However, at sigma-2 receptors, N-phenylpropyl derivatives generally had higher affinity than N-phenethyl derivatives. Furthermore, N-phenylpropyl piperidine and N-phenylpropyl-(2-pyridyl)piperazine exhibited 6-fold and 17-fold selectivity, respectively, for sigma-2 receptors over sigma-1 receptors. Compounds in the N-phenylpropyl series had Ki values of 4.91 - 25 nM at sigma-2 receptors. These compounds will be examined for agonist and antagonist activity. Collaboration continued with Rae Matsumoto (University of Oklahoma) and Amy Newman (National Institute on Drug Abuse) on the role of sigma receptors in cocaine-induced toxicity. Various arylethylene diamine-type sigma ligands (for example, BD1008, BD1063, and LR176) and certain rimcazole analogs (for example SH3/24 and SH2/21) attenuated convulsions induced by cocaine in male Swiss Webster mice. The arylethylene diamines also blocked lethality induced by a dose of cocaine which produced death in 100% of control animals. The potency at these effects correlated with binding affinity at sigma-1 receptors, but not affinity at the dopamine transporter (DAT). These anti-cocaine effects were mimicked by treatment of mice with antisense oligodeoxynucleotides directed against the sigma-1 receptor. These data suggest that antagonism at the sigma-1 receptor is responsible for the blockade of cocaine-induced convulsions and lethality by sigma receptor ligands. The role of sigma receptors in the effects of cocaine will continue to be investigated.